Identification of 2-Aminoacyl-1,3,4-thiadiazoles as Prostaglandin E2 and Leukotriene Biosynthesis Inhibitors

The application of a multi-step scientific workflow revealed an unprecedented class of PGE2/leukotriene biosynthesis inhibitors with in vivo activity. Specifically, starting from a combinatorial virtual library of ∼4.2 × 105 molecules, a small set of compounds was identified for the synthesis. Among these, four novel 2-aminoacyl-1,3,4-thiadiazole derivatives (3, 6, 7, and 9) displayed marked anti-inflammatory properties in vitro by strongly inhibiting PGE2 biosynthesis, with IC50 values in the nanomolar range. The hit compounds also efficiently interfered with leukotriene biosynthesis in cell-based systems and modulated IL-6 and PGE2 biosynthesis in a lipopolysaccharide-stimulated J774A.1 macrophage cell line. The most promising compound 3 showed prominent in vivo anti-inflammatory activity in a mouse model, with efficacy comparable to that of dexamethasone, attenuating zymosan-induced leukocyte migration in mouse peritoneum with considerable modulation of the levels of typical pro-/anti-inflammatory cytokines.

M icrosomal prostaglandin E 2 synthase-1 (mPGES-1), 1 a downstream PG synthase, is a membrane-integrated protein able to convert the cyclooxygenase (COX)-derived unstable prostaglandin H 2 (PGH 2 ) to the bioactive prostaglandin E 2 (PGE 2 ). This enzyme is one of the membraneassociated proteins involved in the metabolism of glutathione and prostanoids (MAPEG), a family of proteins including several key targets, such as the 5-lipoxygenase-activating protein (FLAP), leukotriene C 4 synthase (LTC 4 S), and microsomal glutathione S-transferases, useful for the development of anti-inflammatory and anticancer drugs interfering with prostaglandin and leukotriene biosynthesis. 2 Contrary to the classical non-steroidal anti-inflammatory drugs (NSAIDs), namely blockers of cyclooxygenases (COX-1 and COX-2) and coxibs (COX-2 selective inhibitors), the inhibition of mPGES-1 does not affect the biosynthesis of the other physiologically important PGs. 3,4 Consequently, mPGES-1 inhibitors show a safer profile with respect to fewer gastrointestinal and cardiovascular complications, like thrombosis and vascular inflammation. 5,6 Several studies reported the involvement of this synthase in different types of cancer, 7−9 liver diseases, like viral hepatitis, and drug-induced injury. 10 To date, only two drug candidates are currently in Phase II clinical trials: GRC 27864 is being evaluated for efficacy in patients with osteoarthritic pain; GS-248 is currently being tested in a Phase II trial (https://clinicaltrials.gov/ct2/show/ NCT04744207) in Europe with systemic sclerosis patients (https://clinicaltrials.gov/ct2/results?term=mPGES-1). Thus, the development of mPGES-1 1,10,11 inhibitors represents an urgent issue. Furthermore, in recent years, different series of dual-and/or multi-target inhibitors of eicosanoid biosynthesis targets have been developed. In fact, the use of this type of agents able to block the targets belonging to the three different branches of the arachidonic acid cascade, namely lipoxygenases (LOs), cyclooxygenases (COXs) and cytochrome P 450 monooxygenases (CYP450), may increase the anti-inflammatory effects and reduce the side effects. Indeed, the moderate interference with multiple biological macromolecules may provide advantages in re-adjusting and regulating homeostasis compared to single-target drugs, obtaining the next generation of more efficient and safer anti-inflammatory agents. 12 In the continuous effort to identify mPGES-1 inhibitors, computa-tional tools have always played a central role. 13−15 In this context, considering the broad spectrum of biological activities 16,17 of the 2-amino-thiadiazole derivatives, such as antifungal 18 and antiparasitic activities, 19 and also encouraged by the inhibitory activity shown by 2-aminothiazole-based mPGES-1 inhibitors, 20,21 we investigated the privileged scaffold 2-aminoacyl-1,3,4-thiadiazole as central core for designing potential mPGES-1-blocking agents. To target mPGES-1 protein, in these past few years, we improved and optimized a multi-step computational workflow integrated with robust in vitro, in vivo, and ex vivo experimental analyses 22,23 that allowed us to identify novel dual mPGES-1 and leukotriene biosynthesis inhibitors. Therefore, the generation of a novel library of compounds was the first step in starting our investigation by identifying promising specific chemical platforms for a punctual decoration to be performed according to a selected synthetic approach. Thus, according to the generic scheme reported in Figure 1A, the 2-amino-5-(4bromophenyl)-1,3,4-thiadiazole scaffold was decorated with commercially available acyl chlorides (i.e., 318) and boronic acids (i.e., 570) (Combiglide, LigPrep, QikProp software, see Supporting Information). 24 Considering our final purpose of the in vivo evaluation of the most promising hits, during our investigation we used several computational facilities (Schrodinger Suite 2021) 24 to obtain a small pool of molecules endowed with both favorable pharmacokinetic properties and encouraging pharmacodynamic effects. For these reasons, we first used the LigPrep module to generate all the possible tautomers and protonation states at pH = 7.4 for all molecules belonging to the 2-amino-thiadiazole-based library, obtaining ∼4.2 × 10 5 entities. After that, QikProp and LigFilter software was used to filter out only compounds presenting the wellknown "drug-like" properties. To discard "non-drug-like" compounds and possible false positives in high-throughput screening (HTS) assays, QikProp software 24 was used for the calculation of the pharmacokinetic properties, physically significant descriptors, and pharmaceutically relevant parameters for prediction of absorption, distribution, metabolism, and excretion (ADME). Accordingly, the functional groups generally responsible for reactivity, toxicity, or decomposition problems in vivo were filtered out before the subsequent molecular docking step, in order to rule out "non-drug-like" molecules (Table S1, Supporting Information). Then, the virtual screening workflow (VSW) on mPGES-1 (PDB code: 4BPM) 25 was applied to the final library, containing 1.5 × 10 5 compounds that passed several filters (vide supra), using Glide software. 24 Specifically, the VSW consisted of three subsequent steps, each of them yielding a ranking of compounds according to docking score value: (i) high-throughput virtual screening phase (HTVS); (ii) standard precision phase (SP); and (iii) extra precision phase (XP). The computational analyses of docking results were performed by combining the docking score with a qualitative and visual inspection of a specific set of ligand/mPGES-1 interactions responsible for the inhibitory activity, as already reported by other research groups and by us. 22,26−28 In more detail, considering that a known mPGES-1 inhibitor is able to occupy the peculiar binding groove with a U-shape of the ligand-binding site, in our in silico evaluation the interactions with specific aromatic (i.e., Tyr28 ChainC , Phe44 ChainC , Tyr130 ChainA ), aliphatic (i.e., Val24 ChainC , Val128 ChainA , Leu132 ChainA ), polar (i.e., Pro124 ChainA , Ser127 ChainA , Thr131 ChainA ), and charged (i.e., Arg52 ChainC and Gln134 ChainA ) residues of the pharmacological site of interest were considered for the selection of the best chemical candidates. Specifically, for all the predicted most favored docking poses, the substituents deriving from the side chain of the acyl chlorides preferentially interact with key amino acids, namely Phe44 chainC , His53 chainC , Gly35 ChainC , Asp49 ChainA , and Ser127 ChainA of the cytoplasmic part of the protein ( Figure S1, Supporting Information), while the side chains related to the substitution with the boronic acids ( Figure 1A) establish interactions with the binding pocket (key residues: Val24 ChainC , Tyr28 ChainC , Tyr130 ChainA , Gln134 ChainA , Val 128 ChainA , Thr131 ChainA , and Leu132 ChainA ). Finally, our scientific workflow led to selecting the most promising hits by applying a further filter for excluding the "Pan-Assay Interference compounds" using the SwissADME web tool. 29 This computational tool has allowed a further accurate selection of the best candidates (compounds 1−9, Figure 1B) for (i) chemical synthesis; (ii) biological evaluation in both cell-free and cellbased systems; and (iii) in vivo and ex vivo investigation of the anti-inflammatory properties of the most promising hits.
The novel set of selected compounds targeting mPGES-1 was then synthesized according to the synthetic route reported in Scheme 1. Compounds 1−9 can be divided into groups that differ in some chemical features: (i) compounds bearing the 3hydroxyphenyl moiety (1−4) and (ii) compounds bearing the 2-aminophenyl moiety (5−9). In both cases, the general synthetic route consisted of two main steps: 2-amino-5-(4bromophenyl)-1,3,4-thiadiazole was subjected to Pd-catalyzed Suzuki−Miyaura 30 cross-coupling with 3-hydroxyphenylboronic acid pinacol ester I (line 1, Scheme 1) or 2-(N-Boc- amino)phenylboronic acid II (line 2, Scheme 1) to give 10 and 11, respectively. For the synthesis of these compounds, the Suzuki−Miyaura reaction was performed under standard conditions using [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) as the catalyst and aqueous carbonate as base. The intermediates 10 and 11 were then subjected to an acylation reaction on the amino group of the 2aminothiadiazole ring. Due to the presence of the reactive phenolic group, compound 10 was protected by reaction with trimethylsilyl chloride, and then the proper acyl chlorides were added in situ.
The protecting group was then removed by means of an acid workup of the reaction mixture in order to obtain the final compounds. For compounds 5−9, the tert-butoxycarbonylamino (Boc)-protected boronic acid II was used in the coupling step. The product of the Suzuki reaction was then subjected to acylation with the appropriate acyl chloride, and then the Boc group was removed with a mixture of DCM (50%) and TFA (50%) to give the deprotected aminophenyl derivatives.
Interestingly, like our previous study on aminobenzothiazole derivatives, 13 the 2-bromophenylcarbonyl moiety in compounds 3 and 7 is confirmed to be capable of establishing robust interactions with mPGES-1. From the structural point of view, the biological affinities of compounds 3 and 7 ( Figure  2A and C) could be positively affected by the 2-bromobenzoyl substituent able to accommodate in a deep binding pocket on the mPGES-1 surface. This moiety interacts by van der Waals contacts with amino acids of the cytoplasmic part and establishes π-stacking with Phe44 ChainC and His53 ChainC hydrogen bonds with Ser127, and a halogen bond between the bromine atom and the side chain of Aps49 ChainC . Furthermore, the 3-phenylphenol and 2-aminobiphenyl moieties of 3 and 7 (Figure 2A and C), respectively, are able to establish a peculiar π-stacking with Tyr130 ChainA and a hydrogen bond with Gln134 ChainA . A comparable binding mode is also observed for 6 and 9 ( Figure 2B and D), where also in these cases, the presence of halogen could represent a critical factor in affecting the mPGES-1 activity.
Since compounds 3, 6, 7, and 9 displayed the most potent effects, cell-free assays were performed on several related enzymes involved in the inflammatory response to deeply investigate their anti-inflammatory features and to evaluate their selectivity versus mPGES-1. None of the investigated compounds was active against isolated cyclooxygenases (COX)-1/2, 5-lipoxygenase (5-LO), and soluble epoxide hydrolase (sEH) ( Table 2). Additionally, the effect of compounds 3, 6, 7, and 9 on PGE 2 production was evaluated in a cell-based system, namely in IL-1β-stimulated A549 cells. Compounds 6, 7, and 9 were able to suppress the biosynthesis of PGE 2 in a concentration-dependent manner ( Figure 3A).
Moreover, cell viability assays performed on the A549 cell line and human monocytes excluded that the effects on the levels of PGE 2 were related to possible cytotoxicity of the tested compounds ( Figure 3B, C). Furthermore, the interference of compounds 3, 6, 7, and 9 on the leukotriene biosynthesis was investigated in cell-based systems. Two different experimental settings were applied using intact neutrophils from human peripheral blood: stimulation with Ca 2+ -ionophore or with Ca 2+ -ionophore plus arachidonic acid as exogenous substrate. Interestingly, compounds 3, 6, 7, and 9 inhibited the formation of LTB 4 , its isomers, and 5-H(p)ETE, presenting promising IC 50 values (Table 3 and Figure S3, Supporting Information).  20.9 1.7 ± 0.3 a n = 3; n.d., not determined.

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The absence of 5-LO inhibition in the cell-free assay ( Table  2), in fact, does not exclude an inhibitory activity on FLAP, which is operative in intact cells, and its inhibition by test compounds may explain the impaired cellular LT formation. We confirmed potent inhibition of cellular 5-LO product formation also in human pro-inflammatory macrophages activated by Staphylococcus aureus for 90 min by 3, 6, 7, and 9 but not by 2 (used as negative control), as expected ( Figure  S4, Supporting Information). Notably, the formation of other products derived from 12/15-LOXs was not suppressed but rather elevated. Successively, we sought to investigate the potential inhibitory activity of tested compounds on J774A.1 macrophage stimulated with LPS (10 μg/mL). As shown in Figure 4, compound 3 (1 μM) was able to modulate both IL-6 ( Figure 4A) and PGE 2 ( Figure 4B) production (P ≤ 0.01) in macrophages stimulated with LPS. Compound 7, at the same concentration, displayed a similar activity only in terms of PGE 2 modulation (P ≤ 0.05). The in vitro biological activity assays disclosed compound 3 as the most promising antiinflammatory agent. Therefore, it was selected to evaluate the leukocytes egress into the peritoneal cavity in a mouse model of zymosan-induced peritonitis (see Supporting Information). 32−34 Mice were subjected to intraperitoneal (i.p.) injection of 500 mg/kg zymosan, followed by injection of compound 3. Intraperitoneal injections of PBS alone and of dexamethasone (3 mg/kg) 30 min after zymosan administration were also carried out as an internal control. The strong leucocyte recruitment due to zymosan injection was reduced by administration of compound 3 at the dose of 10 mg/kg at both 4 ( Figure 5A; P ≤ 0.05) and 24 h ( Figure 5B; P ≤ 0.01).
Interestingly, compound 3 showed a remarkable effect even at the lower dose of 1 mg/kg (P ≤ 0.05) at both time points ( Figure 5A, B). Furthermore, leukocyte numbers in the peritoneal cavity at both 4 (P ≤ 0.005; Figure 5A) and 24 h (P ≤ 0.01; Figure 5B) were significantly reduced by dexamethasone. A single administration of zymosan (500 mg/kg) at 4 and 24 h induced a substantial increase in the levels of IL-1β ( Figure 6A, B), IL-6 ( Figure 6C, D), and PGE 2 ( Figure 6E, F) compared to control group. Conversely, a significant reduction of IL-10 levels was observed at both time points ( Figure 6G, H). In addition, IL-1β ( Figure 6A   significantly reduced by compound 3 at 10 mg/kg. The IL-10 level was modulated only at 24 h ( Figure 6H; P ≤ 0.05). Injection of dexamethasone (3 mg/kg) modulated the values of IL-1β, IL-6 PGE 2 , and, conversely, IL-10 with a more prominent effect ( Figure 6). In order to overcome the well-known issue related to the inefficacy of some selective human mPGES-1 inhibitors in mice due to the structural differences in target proteins (i.e., Arg52, which is Lys53 in murine mPGES-1, and His53, which is Arg54 in murine mPGES-1), 35 we also performed homology modeling and molecular docking studies on a murine mPGES-1 model (see Supporting Information).
Specifically, we disclosed the putative interactions of our hits 3, 6, 7, and 9 with the highly conserved region of the active site of murine mPGES-1, ensuring that the 2-amino-1,3,4thiadiazole-based compounds could similarly bind both the isoforms, showing comparable binding modes in the active sites of the enzymes (see Supporting Information and Figure  S6). In line with the widespread presence of thiadiazole in the chemical structure of several pharmacologically active com-

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pubs.acs.org/acsmedchemlett Letter pounds, we here demonstrated that 2-amino-1,3,4-thiadiazolebased compounds, when opportunely decorated, are promising anti-inflammatory hits. The application of a multi-step computational protocol, based on a concise synthetic method, allowed us to single out diverse substituted 2-acylamino thiadiazole derivatives, which led to the identification of the novel hits 3, 6, 7, and 9 as potent PGE 2 biosynthesis inhibitors, with 3 and 7 showing the strongest effects. In addition, the compounds were screened against several enzymes involved in the inflammatory response using cell-free assays. First, no activity was found against COX-2 as well as the constitutively expressed COX-1, ensuring the absence of the well-known side effects due to the action on COX targets (vide supra), making these compounds interesting candidates as safer alternatives, especially for long-term therapies.
Moreover, the compounds were not able to interfere with 5-LO activity in cell-free assays, while their ability to strongly interfere with cellular biosynthesis of leukotrienes was demonstrated, presumably due to interference with FLAP. Finally, in vivo and ex vivo results also demonstrated that the zymosan-induced leukocyte migration was attenuated by treatment with compound 3, with a significant modulation in the levels of typical pro-/anti-inflammatory cytokines with efficacy similar to that of dexamethasone. Finally, we performed homology modeling and molecular docking toward the murine mPGES-1 to corroborate the potential utility of this molecular scaffold as a modulator of the PGE 2 level in both murine and human models.
In summary, our multidisciplinary workflow, which combines in silico studies, chemical synthesis, and cell-free  and cell-based assays, represents a fast and powerful method for identifying novel hits that are able to inhibit PGE 2 and LT biosynthesis with in vivo anti-inflammatory activity. ■ ASSOCIATED CONTENT * sı Supporting Information of mPGES-1 inhibitors suitable for preclinical testing in wild-type mice as a new generation of anti-inflammatory drugs. Sci. Rep. 2018, 8, 5205.